Twentieth century blood testing work has evolved into separate, specialized areas such as:
Coagulation PA1 Hematology PA1 Blood chemistries and enzymes PA1 Electrolytes and trace metals PA1 Serology PA1 (1) antithrombins, such as heparin, PA1 (2) those that remove calcium ions from the blood to prevent coagulation, such as citrate, oxalate, fluoride, and ethylene diamine tetra acetic acid (EDTA).
Present techniques for blood analysis require the use of different anticoagulants for different analyses of in vitro blood samples. These anticoagulants are chosen for optimum effect in specific uses, and are seldom interchangeable.
The in vitro anticoagulants in use at present are of two types:
In coagulation studies, calcium ion must be prevented from initiating a coagulation reaction in freshly drawn blood samples. Plasma is separated from red cells and then recalcified under controlled conditions. The most commonly used anticoagulant for in vitro coagulation studies is citric acid. It is generally employed in the form of sodium citrate. EDTA is not used in coagulation procedures because abnormally high coagulation times are obtained upon recalcification of EDTA plasmas. Oxalate, fluoride, heparinized plasma, and serum are of little or no use in coagulation studies.
Ethylene diamine tetra acetic acid (EDTA) is the preferred in vitro anticoagulant for hematology studies. It is usually employed in the form of the sodium or potassium salt. Sodium citrate is not used in hematology procedures because cell morphology is altered, and some platelets are destroyed in citrated blood. Unanticoagulated whole blood may be used for making stained blood smears for microscropic examination of cell morphology; heparin may be used as an anticoagulant for all hematological tests except morphology, because anionic heparin reacts with the cationic dye used for staining blood smears. Fluoride and oxalate have limited use.
The chief requirement for a fluid medium for testing in the areas of blood chemistries, enzymes, electrolytes, and trace metals is that the test medium contain no ingredient that will interfere with the tests being conducted. Serum or heparinized plasma are the desired media. However, serum potassium values are often high, due to potassium released into serum by platelets and red cells during the coagulation process; heparin plasmas often yield false high phosphorus results, and bring about enzyme inhibition.
Citrated of EDTA plasmas often result in errors in calcium determinations and alkaline phosphatase determinations. Oxalate and fluoride anticoagulants also result in interference in many of these determinations.
It is an object of this invention to provide an in vitro anticoagulant which can be used in coagulation and hematology determinations, but which will not interfere with chemistries, enzymes, electrolytes or trace metals.
The stability constant, K, for a metal chelate is the ratio of chelated metal ions in a solution at equilibrium to that of unchelated metal ions. For calcium chelates, the stability constant may be expressed as follows: ##EQU1## Since K is usually a very high number, log K is the favored expression in the technical literature. Stability constants for the calcium chelates under consideration are:
______________________________________ Citric Acid log K = 3.2 EDTA log K = 10.7 ______________________________________
Coagulation times increase as the value of K increases. In the art of blood testing work, it is common to encounter the symbol K', the conditional calcium salt stability at pH 7.5. The pH value of 7.5 is the pH level of blood plasma at 25.degree. C. As coagulation times increase, the value of K', like the value of K, increases. A chelate having a log K' of 3 is sufficiently low in stability to permit plasma recalcification for coagulation studies, but not sufficiently high in stability to permit hematological studies. A chelate having a log K' appreciably higher than 4 is sufficiently high in stability to permit use in hematological studies, but not sufficiently low in stability to permit recalcification for coagulation studies.
I have discovered that the use of certain compounds having calcium salt stability constant of log K' of about 3.4 to about 4.2, preferably about 4, namely N-(2-acetamido)-imino diacetic acid, and hydroxyethyl imino diacetic acid, and alkali metal or alkaline buffer salts of these compounds will permit dual use as an in vitro anticoagulant in coagulation studies and in hematology studies. The alkali metal salts that are suitable in this invention are potassium, sodium, and lithium salts. The alkaline buffer salts that are suitable are Imidazole and Tris salts.
At calcium salt stability of log K' of 4, these selected compounds yield coagulation results similar to those yielded by conventional sodium citrate anticoagulants. The compounds also yield hematological results similar to those yielded by conventional EDTA anticoagulants.
Faust et al., U.S. Pat. No. 2,193,717 claims a process for preventing the congealing of blood by using a water-soluble amino acid containing in the molecule more than one organic radical having a carboxylic group on each basic nitrogen atom; more specifically, it calls for the use of either the sodium salt of nitrilo-triacetic acid or the sodium salt of ethylene-bis (imino-diacetic acid) (EDTA).
EDTA will not permit the coagulation testing that the chelates of my invention will permit. EDTA is effective for hematology studies, but is undesirable for coagulation studies. Nitrilo-triacetic acid, although rarely used as an in vitro anticoagulant, is effective for hematology studies, but is ineffective for coagulation studies.
At the present time, a minimum of three blood samples is required for a complete patient profile in coagulation, hematology, and blood chemistry. A citrated sample is required for coagulation, an EDTA anticoagulated sample is required for hematology, and an unanticoagulated sample is required to obtain serum for blood chemistry. Although volumes of blood drawn for the first two samples are usually of 5 ml or 7 ml, only a few tenths of a milliliter is used for testing. This procedure is merely wasteful for robust donors who are sampled infrequently, but it presents a serious problem in obtaining valid samples from pediatric or geriatric patients, who often must be tested frequently.
Frequent, repeated sampling of the pediatric or geriatric patient often results in partial collapse of the vein during blood sample withdrawal, and consequent hemolysis of blood samples. Hemolyzed EDTA blood samples may be used for most hematology testing, with the exception of red cell fragility; hemolyzed serum samples will give falsely high readings from iron, potassium, triglycerides, transaminase enzymes, and lactate dehydrogenase; hemolyzed citrated samples are worthless for coagulation studies.